Synthesis And Properties Invesigation Of Cationic Polymer Gene Carrier

Studies have proved that many human diseases are closely related with the changeof gene structure or its function, gene therapy has become an emerging medicaltreatment to improve human health. The key of gene therapy lies in the choice of theappropriate gene carrier. Developing a kind of gene vector that is biological degradingwith high transfection efficiency, low toxicity and targeting specificity has become amajor task at present in this field.Because of its “proton sponge effect”, Polyethylenimine (PEI), a gene vector withhigh efficiency has become the research hotspot. Its structure determines that it canboth carry and release a great deal of genes, but its cell toxicity limits its application inorganism. Chitosan (CS) shows low toxicity and high biological compatibility matchedby no other cationic polymer vector, but low transfection efficiency, Its specialmolecular structure determines its poor solubility in neutral or physiological pHenvironment. Polyethylene glycol (PEG) prezents good biocompatibilit y, a number ofwater molecules can be combined with the hydrophilic chains to nanoparticles, thatimprove other water-soluble polymer material increase in blood circulation time in thesystem.Based on the PEG with good biocompatibility, PEI with strong ability to bindDNA and high transfection efficiency, and CS with low toxicity and high biologicalcompatibility matched by no other cationic polymer vector, a new original andbiodegradable cationic polymer gene carrier (PA5) was designed and synthesized. Thispolymer should have good hydrophilicity, low or no toxicity, high gene transfectionefficiency and good biological compatibility. This new vector takes the advantages andavoids the disadvantages of PEG, PEI and CS. In this thesis, the characterization ofvarious biological properties shows that PA5, the new and original cationic polymer,has high transfection, lower toxicity and good biological performance. The followingworks were finished mainly in this thesis:(1) Synthesis and characterization of copolymer of polyethylene glycol andpolyethylene imine–chitosan (PA5)A new original and biodegradable gene vector PA5was synthesized. Under theprotection of the boric acid, hydrophilic PEG is modified into double functionalcompounds HO-PEG-COOH (PA3), which provides a new method for PEG modification. The water solubility of chitosan was increased greatly and its reactivitywas improved by carboxylation reaction of chitosan. The new and original copolymerPA5was prepared through the link of the modified PEG, CS and PEI and PA5wascharacterized with IR and1H NMR. With its amphiphilic characteristics, thecopolymer PA5can be self-assembled to form nanoparticles with core-shell structurein aqueous solution.(2) Synthesis and characterization of chitosan-grafted copolymer carrier (PB2)Using chitosan as starting materails, the non-toxic and biocompatible copolymercarrier (PB2) was designed and synthesised. In the prensence of acetonitrile potassium,HO-PEG-CN was prepared by ring-opening polymerization of ethylene oxide. Andthen HO-PEG-CN was hydrolyzed to HO-PEG-COOH (PA2). PA2reacted with PA4tosynthesize PB1through amidation reaction, and then PB1reacted withp-aminobenzaldehyde given PB2in in nearly quantitative yield. The product can directbe used in the next step reaction without purification.This amphiphilic copolymer,containing an aldehyde group and a considerable amount of amine, can be used asgene/drug common transmission carrier.(3) Bioactivities evaluation of Copolymer carrier PA5The Bioactivities of PA5as a gene vector was evaluated. The binding capacity ofpolymers PA5with EGFP-N1type of DNA was confirmed by agarose gelelectrophoresis, when the mole ratio of PA5and DNA is six the DNA in the system iscompletely binded by PA5. The cytotoxicity of different polymers has been determinedby MTT method. The cytotoxicity of the PA5to HeLa was grade1(C<50ug/mL), betterthan PEI. The optimum conditions of transfected Hela cells in the presence of serumwere studied: the composite time of PA5/DNA is30min, the time of transfected Helacells is6hours and the best transfection efficiency was48.2%. The transfectionefficiency of PA5in10%serum is evidently higher than that under non-serumcircumstance, illustrating the polymer PA5keeps the advantages of the CS.(4) Synthesis, characterization and bioactivities evaluation of Poly aminobutylene lipid (PC2).Polyaspartic acid butanediol ester (PC2) was synthesized with butylene glycol andaspartic acid as substrate and its feasibility of biomedical material application wasstudied. PC2degrades in both Novozym435enzyme buffer solution and simulatedbody fluid environment. The cytotoxicity to L929cells was grade0. The bindingcapacity of polymers PC2with DNA was confirmed by agarose gel electrophoresis,when the mole ratio of PA5and DNA is8the DNA in the system is completely binded by PC2. The polymer can be applied in targeting drug reducing carrier andtissue-engineered scaffold material.